1. 1

2. Part I: Humble beginnings
Outline
tracrRNA
CRISPR
Genomic DNA
cas9
cas1
cas2
csn2
Part I: Humble beginnings
Part II: Harnessing CRISPR
Part III: Lessons of CRISPR 2

3. Part I: Humble beginnings3

4. First report about transformation
O’Connor C., 2008, Nature Education

5. First report about transformation
Experiment
Result S S R R S
Dead S + R
Something inside of S type bacteria induced “R--->S” transformation
Griffith Fred, 1928, J. Hygiene

6. DNA could be a “Gene” Experiment Result S S S R R R R DNA Protein
Lipid R
Carbohydrate R
Avery O. T., 1944, J. Expt. Med.

7. The “Gene” is DNA only
1969, Max Delbruck, Salvador E. Luria, and Alfred Hershey share the Nobel Prize in Physiology and Medicine
Hershey A. D. and Chase M., 1952, J. General Physiology.

8. And then… Dr. Max Delbrück Dr. Alfred Hershey Dr. Salvador Luria
Discoveries the genetic structure of viruses in 1952
Dr. Kary Banks Mullis
Invent Polymerase chain reaction (PCR) in 1983
Dr. Frederick Sanger
Contributions in DNA sequencing in 1955
Dr. James Watson and Dr. Fredrick Crick
Describe structure of DNA in 1953
Dr. Arthur Kornberg
Discover the enzyme DNA polymerase I in 1956
Hershey and chaseThe Nobel Prize in Physiology or Medicine 1969
"for their discoveries concerning the replication mechanism and the genetic structure of viruses“
Watson and CrickT he Nobel Prize in Physiology or Medicine 1962
ΦX174) bacteriophage was the first DNA-based genome to be sequenced. This work was completed by Fred Sanger and his team in 1977.The Nobel Prize in Chemistry 1980, "for their contributions concerning the determination of base sequences in nucleic acids"
In 1956, Arthur Kornberg and colleagues discovered the enzyme DNA polymerase I, also known as Pol I, in Escherichia coli.
The Nobel Prize in Physiology or Medicine 1959,  "for their discovery of the mechanisms in the biological synthesis of ribonucleic acid and deoxyribonucleic acid"
Mullis The Nobel Prize in Chemistry 1993

9. Bacteriophage
Humans are not alone in having to fend off pathogens; even the simplest organisms are under a constant threat of invasion. Bacteria, for example, are awash in a sea of viruses known as bacteriophages. “Every 2 days, half the bacteria on Earth are killed [by bacteriophages],”
RNA sequencing was one of the earliest forms of nucleotide sequencing. The major landmark of RNA sequencing is the sequence of the first complete gene and the complete genome of Bacteriophage MS2,
The first full DNA genome to be sequenced was that of bacteriophage φX174 in 1977, 5386bp

10. short palindromic sequences

11. Identification of CRISPR
Spacer
we will refer to this family as the clustered regularly interspaced short palindromic
repeats (CRISPR).
The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four
CRISPR-associated (cas ) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes.
Rodolphe Barrangou and Philippe Horvath were working at Danisco, a yogurt company, and they were curious about strategies to help their yogurt cultures survive viral infections. They started infecting their bacteria with viruses and looked for viral DNA in the CRISPR spacers. Sure enough, the viral DNA was contained in the immune cells. When the DNA sequences were removed from the spacers, immunity was lost. In 2007, there was mechanistic proof that CRISPR served as a mechanism to defend against viruses using a completely novel mechanism of action. 
Clustered Regularly-Interspaced Short Palindromic Repeats
CRISPR

12. Identification of cas genes
Addison V. Wright, et al. 2016, Cell
two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein
complexes, whereas class 2 effectors rely on single-component effector proteins such as the wellcharacterized
Cas9.
we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR).
The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four
CRISPR-associated (cas ) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes.
Rodolphe Barrangou and Philippe Horvath were working at Danisco, a yogurt company, and they were curious about strategies to help their yogurt cultures survive viral infections. They started infecting their bacteria with viruses and looked for viral DNA in the CRISPR spacers. Sure enough, the viral DNA was contained in the immune cells. When the DNA sequences were removed from the spacers, immunity was lost. In 2007, there was mechanistic proof that CRISPR served as a mechanism to defend against viruses using a completely novel mechanism of action. 
Cas, CRISPR associated proteins

13. CRISPR BLAST
Nature rejected it In November 2003: without seeking external review;
PNAS rejected it in January 2004:r lacked sufficient ‘‘novelty and importance’’
Molecular Microbiology rejected…
Nucleic Acid Research rejected the paper in turn...
Bolotin was the first to speculate how CRISPR conferred immunity—proposing that transcripts
from the CRISPR locus worked by anti-sense RNA inhibition of
phage gene expression. Although reasonable, the guess wouldprove to be wrong.

14. RNAi-like CRISPR-Cas system (CASS)
we will refer to this family as the clustered regularly interspaced short palindromic
repeats (CRISPR).
The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four
CRISPR-associated (cas ) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes.
Rodolphe Barrangou and Philippe Horvath were working at Danisco, a yogurt company, and they were curious about strategies to help their yogurt cultures survive viral infections. They started infecting their bacteria with viruses and looked for viral DNA in the CRISPR spacers. Sure enough, the viral DNA was contained in the immune cells. When the DNA sequences were removed from the spacers, immunity was lost. In 2007, there was mechanistic proof that CRISPR served as a mechanism to defend against viruses using a completely novel mechanism of action. 
Pourcel C, et al., Microbiology, 2005, 151(Part 3):
Alexander B, et al. Microbiology, 2005, 151(Part 8):
Makarova K S, et al. Biology Direct, 2006, 1(1):1-26.

15. CRISPR confers adaptive immunity
By late 2004, he noticed a clear correlation between spacers and phage resistance—as would be reported just a
few months later by Mojica and Vergnaud.

16. Programming CRISPR
Complementary to:
RNA and coding strand of DNA
Template strand
Cas3, one of the Cas proteins in E. coli K12 CRISPR/cas system
Cascade, casABCDE, CRISPR-associated complex for antiviral defense
Host sensitivity to phages was tested using a virulent variant of phage Lambda (lvir)
First case of directly programming CRISPR-based immunity—a flu shot for bacteria.
Brouns S J J, et al., Science, 2008, 321(5891):

17. CRISPR Targets DNA Plasmid Plasmid
Self-splicing intron
“From a practical standpoint, … it’s possible to manipulate genomic DNA”
- Marraffini and Sontheimer
Marraffini L A and Sontheimer E J. Science, 2009, 322(5909): 1843–1845.

18. Discovery of tracrRNA
he approach yielded a striking result: the third-most abundant class of transcript—after only ribosomal RNA and transfer
RNA—was a novel small RNA that was transcribed from a sequence immediately adjacent to the CRISPR locus (in the
region that had caught Charpentier’s attention) and had 25 bases of near-perfect complementary to the CRISPR repeats.
Model for tracrRNA-mediated crRNA maturation involving RNase III and Csn1.
Black, repeat; green, spacer. tracrRNA can bind with almost perfect complementarity to each repeat sequence of the pre-crRNA. The resulting RNA duplex is recognized and site-specifically diced by RNase III in the presence of Csn1, releasing the individual repeat-spacer-repeat units (first processing event). The generated units undergo further processing within the spacer sequences resulting in mature crRNA species consisting of unique spacer-repeat sequences (second processing event) by a yet-to-be elucidated mechanism. Csn1 may also be involved in.
tracrRNA was not only involved in processing crRNA but was also essential
for the Cas9 nuclease complex to cleave DNAthe silencing of invading sequences.
tracrRNA, trans-activating CRISPR RNA

19. Flexibility in CRISPR processing systems
In CRISPR arrays of DNA, conserved repeats (squares) alternate with variable spacers (diamonds). During the acquisition/memory step, new spacers are incorporated as a result of invasion of the cell by a phage or plasmid. During the processing steps required to confer immunity, RNA transcripts of the CRISPR array are cleaved to give mature guide RNAs, which then target for destruction invading genomes that match the spacer. The related proteins CasE, Csy4 and Cas6 carry out the initial processing step in many organisms. Deltcheva et al.2 describe a different pathway for guide-RNA maturation that operates in Streptococcus pyogenes and certain other bacteria (boxed area). This pathway involves a trans-acting RNA (tracrRNA) that hybridizes with the spacers, leading to cleavage by RNase III. Csn1 aids in the process, and an undefined step yields mature guide RNAs.
Susan G. 2011, Nature, 471(7340):

20. Structures of Cas proteins
Csy4 bound to RNA substrate
CasA
The CASCADE complex
Martin J, et al. 2014, Science, (6176): ; Wiedenheft B, et al., 2009, Structure, 17(6): ; Haurwitz R E, et al. 2010, Science, 329(5997):

21. Emmanuelle Charpentier, Jennifer Doudna, Martin Jinek, Krzysztof Chylinski and Ines Fonfara

22. Flexibility in CRISPR processing systems

23. Each Cas9 nuclease domain cleaves one DNA strand
Incubation of these variant Cas9 proteins with native plasmid DNA showed
that dual-RNA–guided mutant Cas9 proteins yielded nicked open circular plasmids, whereas
the WT Cas9 protein-tracrRNA:crRNA complex produced a linear DNA product
Cas9 uses two nuclease domains to cleave the two strands in the target DNA

24. Cleavage sites in target DNA
PAM, protospacer adjacent motif
tracrRNA might be required for targetDNAbinding and/or to stimulate the nuclease activity of Cas9 downstream of
target recognition. To distinguish between these possibilities, we used an electrophoretic mobility
shift assay tomonitor target DNA binding by catalytically inactive Cas9 in the presence or absence of crRNA and/or tracrRNA.
Minimal regions of functional tracrRNA and crRNA

25. Secondary structure of Dual-tracrRNA: crRNA
S. pyogenes
Dual-tracrRNA: crRNA guided target DNA cleavage by Cas9 is species specific
L. innocua
N. meningitidis
Martin Jinek, etc., 2012, Science (Suppl.)

26. Cas9 can be programmed with sgRNA

27. CRISPR/Cas9 system

28. Crystal Structure of Cas9/gRNA Complex and Target DNA
This high-resolution structure and accompanying functional analyses have revealed the molecular
mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new,
versatile genome-editing technologies.
Hiroshi N., et al., Feb, 2014, Cell, 156(5):935–949

29. Structures of Cas proteins
Upon binding the crRNA:tracrRNA guide, the two structural lobes of Cas9 reorient
such that the two nucleic acid binding clefts face each other. This generates a central DNA binding
channel, which allows access to dsDNA. Target DNA binding in the central channel and PAM-dependent
R-loop formation result in a further structural rearrangement. Here, the nuclease domain lobe undergoes
further rotation relative to the a-helical lobe, fully enclosing the DNA target, and the two nuclease
domains engage both DNA strands for cleavage.
Martin J, et al. Mar, 2014, Science, (6176): ;

30. PAM recognition regulates Cas9 nuclease activity
Model for target search, recognition and cleavage by Cas9–RNA. The search initiates through random three-dimensional collisions. Cas9–RNA rapidly dissociates from non-PAM DNA, but binds PAMs for longer times and samples adjacent DNA for guide RNA complementarity, giving rise to a heterogeneous population of intermediates. At correct targets, Cas9–RNA initiates formation of an RNA–DNA heteroduplex, and R-loop expansion propagates via sequential unwinding. The DNA is cleaved, and Cas9–RNA remains bound to the cleaved products.
Sternberg S H, et al. March, 2014, Nature, 507(7490):62-67.

31. Twenty-Year story of CRISPR
Figure 2. The Twenty-Year Story of CRISPR Unfolded across Twelve Cities in Nine Countries
For each ‘‘chapter’’ in the CRISPR ‘‘story,’’ the map shows the sites where the primary work occurred and the first submission dates of the papers. Green circles refer to the early discovery of the CRISPR system and its function; red to the genetic, molecular biological, and biochemical characterization; and blue to the final step of biological engineering to enable genome editing.
Eric S. Lander, 2016, Cell

32. Part II: Harnessing CRISPR
Huntington’s disease, Sickle-cell anemia, diabetes, autism, AD, cancer
The cancer genome atlas, sequencing 10 million mutations related to cancer.
We demonstrate CRISPR-Cas9–mediated correction of a Fah mutation in hepatocytes in a mouse model of
the human disease hereditary tyrosinemia. is possible in adult animals and has potential for correction of human
genetic diseases. 32

33. DNA sequence disrupted
NHEJ and HDR
DNA
DNA sequence disrupted
NHEJ: Non-homologous end joining
+donor DNA
DNA sequence replaced
HDR: homology directed repair

34. ZFN: Zinc finger nuclease
ZFN and TALEN
Jeffrey C Miller, et al. 2007, Nature Biotechnology, 25, 778 – 785.
ZFN: Zinc finger nuclease
Like cruise missiles attacking military targets, these proteins, called transcription
activator–like effectors (TALEs), can be programmed to home in on specific DNA
sequences and carry out an action once there.
Each TALE repeat targets a specifi c DNA base: A, T, C, or G. The repeats are the same series of 34 animo acids (color-coded) except at positions 12 and 13, which differ depending on the base being targeted.
Once he had the code in hand, Bogdanove immediately wondered if TALEs could
replace zinc fi ngers as the targeting mechanism for nucleases—creating so-called
TALENs.
Elizabeth P. , 2012, Science
TALEN: transcription activator–like effector-based nuclease

35. Genome Editing in Mammalian Cells
I: Genome modification
II: Genomic deletion
Le C, F Ann R, David C, et al. 2013, Science, (6121):

36. Models generated by CRISPR/Cas9 system
Dumpier nematodes
Zebrafish embryos
Fruit flies
Monkey
Rice
Pennisi E Science, (6148):833-6.

37. “Off-target" effect High efficiency (48.3%) Low efficiency (14.3%)
Liang, P., et al., 2015, Protein Cell, 6(5): (Received March 30, 2015 Accepted April 1, 2015)

38. Cpf1, a Single RNA-Guided Endonuclease
Cpf1, CRISPR from Prevotella and Francisella 1
Various strategies have been described to reduce genome-wide off-target mutations of
the commonly used SpCas9 nuclease, including: truncated sgRNAs bearing shortened regions of target site complementarity, SpCas9 mutants such as the recently described D1135E variant, paired SpCas9
nickases, and dimeric fusions of catalytically inactive SpCas9 to a non-specific FokI nuclease.
Now, Zetsche et al. establish a previously
uncharacterized CRISPR−Cas protein, Cpf1, as a novel tool with several advantages over Cas9. First, whereas Cas9 generates
cleavage products with blunt ends, Cpf1 makes staggered cuts, resulting in a 5 ʹ overhang that improves the precision of DNA
insertions. Second, unlike Cas9, Cpf1 cuts at a distal site, which preserves the seed region — essential for target recognition — for future editing. Third, the T-rich protospacer-adjustment motif (PAM; a secondary recognition site) makes Cpf1 better suited to editing AT-rich DNA than Cas9, which has a G-rich PAM. Last, Cpf1 may be easier to deliver to cells, as it is smaller and does not require a tracrRNA
Cpf1 makes staggered cuts
Cpf1 cuts at a distal site
T-rich PAM
Cpf1 may be easier to deliver to cells
Zetsche B, et al., Cell, Oct. 2015, 163(3):
Fagerlund R D, et al., Genome Biology, 2015, 16(1):1-3

39. SpCas9-HF1
The majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1
Benjamin P. Kleinsiver, et al., Nature, 2016, Received 08 November 2015, Accepted 09 December 2015

40. Pop in and out
Two step, ‘pop in & out’ approach for the generation of CRISPR-Cas9-induced targeted mutants. a In the ‘in’ step, the targeting vector (homology regions shaded gray) introduces a tag segment that is disrupted by a loxP-flanked green fluorescent protein (GFP) reporter gene. A Cas9 and sgRNA-induced double strand break (DSB) stimulates homology-directed repair (HDR) and enables the enrichment of targeted GFP+ cells by fluorescence-activated cell sorting (FACS). In the ‘out’ step, the marker is deleted by Cre/lox-mediated recombination and GFP− cells are subsequently enriched by FACS. b Two step in & out targeting approach for the seamless removal of a marker gene. In the first step, the targeting vector introduces a nucleotide replacement (a single nucleotide polymorphism (SNP)) next to a GFP reporter. The marker is removed from the targeted allele using Cas9 and a pair of sgRNAs that recognize the end of the marker cassette. The marker gene is removed by HDR with a targeting vector to provide sequences (homology regions shaded) that are wild type except for the SNP, and GFP− cells are subsequently enriched by FACS

41. Utilization of CRISPR/Cas9 system
Hsu, P. D., et al., 2014, Cell 157(6):

42. Part III: Lessons of CRISPR42

43. funding agencies, the general public, and aspiring researchers.
The story of CRISPR is rich with lessons about the human ecosystem that produces scientific advances, with relevance to
funding agencies, the general public, and aspiring researchers.
The most important is that medical breakthroughs often emerge
The discovery of the CRISPR loci, their biological function, and the tracrRNA
all emerged not from wet-bench experiments but from open-ended bioinformatic exploration of large-scale, often public,
genomic datasets. from completely unpredictable origins.
Ledford H. Nature, 2015, 522(7554):20-4.

44. This year, several leading researchers have sounded warnings about the risks of using the CRISPR gene-editing technique to modify human and other species’ genomes in ways that could have “unpredictable effects on future generations”and “profound implications for our relationship to nature”
The never-ending debates about genetically modified (GM) organisms, nuclear power, chemical toxicity and the efficacy of cancer screening should be evidence enough that science does not limit or resolve controversies about risk.
Democratically weighing up the benefits and risks of gene editing and artificial intelligence is a political endeavor, not an academic one.
--Daniel Sarewitz.

45. Collaborations on CRISPR/Cas9 system
国拜耳宣布投资3亿美金与初创基因编辑公司CRISPR Therapeutics合作开发新药用于血液疾病、失明和先天性心脏病的治疗，这也仅仅只是制药公司渴求通过CRISPR技术开发新药的最初迹象。
In the second deal of its kind, Juno Therapeutics (NASDAQ: JUNO) of Seattle has tapped CRISPR-Cas9 experts Editas Medicine, of Cambridge, MA, to help engineer the T cells of cancer patients into more efficient cancer killers. They will work together on three undisclosed research programs in both branches of Juno’s cancer immunotherapy work, known in shorthand as CAR-T and TCR therapies.
 Novartis was the first to marry CRISPR-Cas9 and CAR-T
the therapeutic applications of CRISPR
advances in genome editing technology
challenges to progressing disease models
CRISPR-based genome-wide screening
DNA repair
the biosafety of gene editing

46. Acknowledgment Dr. Su Qian Dr. Erich Grimm Xiaojian Wang Kun Liu
Kun, up to today, he sent me 210 s, where 180 s with attachment, 412 literatures in total.
Acknowledgment
Dr. Su Qian
Dr. Erich Grimm
Xiaojian Wang
Kun Liu
Lilly Xie